Sequence analysis of the tetravalent rotavirus vaccine

ABSTRACT

Isolated nucleic acid molecules comprising a gene segment from a rhesus rotavirus (RRV) or from one of three rhesus:human reassortant viruses are disclosed, including isolated nucleic acid molecules having a sequence selected from the group consisting of: SEQ ID NO: 1-14, inclusive, and isolated nucleic acid molecules encoding a protein having a sequence selected from the group consisting of SEQ ID NO: 15-28, inclusive, as well as variants of the isolated nucleic acid molecules.

RELATED APPLICATION

This application claims the benefit of U.S. Application No. 60/359,960, filed Feb. 27, 2002. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Rotavirus is a segmented, double-stranded RNA virus that is the major cause of severe gastroenteritis in infants and young children. Profound fluid and electrolyte loss often leads to hospitalization for life-saving rehydration therapy. Where access to medical care is limited or unavailable, volume depletion, shock and death can occur. Annually, nearly one million childhood deaths in developing countries have been ascribed to inadequately treated rotavirus gastroenteritis. In the industrialized world, more than 30% of the children admitted to hospitals with acute gastroenteritis have rotavirus infections.

SUMMARY OF THE INVENTION

The invention is drawn to isolated nucleic acid molecules comprising a gene segment from a rhesus rotavirus (RRV) or from one of three rhesus:human reassortant viruses. In one embodiment, the isolated nucleic acid molecule has a sequence selected from the group consisting of: SEQ ID NO: 1-14, inclusive. In another embodiment, the isolated nucleic acid molecule encodes a protein having a sequence selected from the group consisting of: SEQ ID NO: 15-28, inclusive. In yet another embodiment, the isolated nucleic acid molecule is a variant of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, or 14, such as one of the following: a nucleic acid molecule having the sequence of SEQ ID NO: 1 (gene 1) and having a nucleotide change to a G at nucleotide 2120; a nucleic acid molecule having the sequence of SEQ ID NO: 2 (gene 2) and having one or more of the following nucleotide changes: a C at nucleotide 493, and a T at nucleotide 947; a nucleic acid molecule having the sequence of SEQ ID NO:3 (gene 3) and having one or more of the following nucleotide changes: an A at nucleotide 169, a C at nucleotide 283, a C at nucleotide 448, a C at nucleotide 874, a C at nucleotide 1306, and a C at nucleotide 2388; a nucleic acid molecule having the sequence of SEQ ID NO:4 (gene 4) and having one or more of the following nucleotide changes: a C at nucleotide 119, a G at nucleotide 417, a G at nucleotide 809, a C at nucleotide 977, an A at nucleotide 1463, a C at nucleotide 1481, a C at nucleotide 1608, a C at nucleotide 1755, and an A at nucleotide 1953; a nucleic acid molecule having the sequence of SEQ ID NO:5 (gene 5) and having one or more of the following nucleotide changes: an A at nucleotide 75, a T at nucleotide 84, a C at nucleotide 347, a T at nucleotide 667, a C at nucleotide 1186, a G at nucleotide 1219, and an A at nucleotide 1204; a nucleic acid molecule having the sequence of SEQ ID NO: 6 (gene 6) and having one or more of the following nucleotide changes: a C at nucleotide 376, an A at nucleotide 756, an A at nucleotide 1008, and a G at nucleotide 1041; a nucleic acid molecule having the sequence of SEQ ID NO: 7 (gene 7) and having a nucleotide change to a G at nucleotide 387; a nucleic acid molecule having the sequence of SEQ ID NO:10 (gene 10) and having one or more of the following nucleotide changes: an A at nucleotide 92, an A at nucleotide 174, and a G at nucleotide 218; a nucleic acid molecule having the sequence of SEQ ID NO: 11 (gene 11) and having a nucleotide change to A at nucleotide 180; a nucleic acid molecule having the sequence of SEQ ID NO: 12 (DxRRV (serotype 1)) and having a nucleotide change to an A at nucleotide 556; and a nucleic acid molecule having the sequence of SEQ ID NO: 14 (ST3xRRV (serotype 4)) and having a nucleotide change to a G at nucleotide 263. Each gene variant can have one, more than one, or all of the nucleotide changes enumerated for that particular genie. Other variants of any one of nucleic acid molecules having SEQ ID NO:1-14, or encoding a polypeptide of SEQ ID NO: 15-28, are also included.

DETAILED DESCRIPTION OF THE INVENTION

Human rotavirus serotypes G1-G4 are the major causes of diarrheal gastroenteritis in humans (Gentsch, et al., 1995). The serotypes are determined by epitopic differences in the outer capsid of the virus particle encoded by the VP7 gene. The ROTAMUNE™ (ROTASHIELD™) vaccine is a live virus vaccine comprised of four different rotaviruses, each containing the outer capsid protein, VP7, of one of the major serotypes G1-G4 known to cause disease in humans. The foundation of this vaccine is a virus isolated from a rhesus macaque, rhesus rotavirus (RRV). The virus is sufficiently similar to human strains to permit limited replication in human intestinal tracts and thereby elicit protective immune responses to human rotaviruses. The ROTAMUNE™ vaccine includes RRV (serotype G3, for which VP7 is 96% homologous to VP7 from human serotype 3 viruses); and three rhesus:human reassortant viruses (serotypes G1, G2 and G4). The reassortants are comprised of the rhesus virus genetic background (10 gene segments), but replace the gene segment encoding VP7 with the corresponding gene segments from the human serotype 1 (D strain), 2 (DS 1 strain) or 4 (ST3 strain) viruses. Applicants have, for the first time, identified the nucleic acid sequence of all 11 gene segments of each of the four virus strains, including the 10 common gene segments and the four independent gene segments (VP7 gene), for a total of 14 gene segments.

Nucleic Acids of the Invention

Accordingly, the invention pertains to an isolated nucleic acid molecule comprising a gene segment from the rhesus rotavirus (RRV) or from one of the three rhesus:human reassortant viruses. The term, “gene segment,” as used herein, refers to a nucleotide sequence, preferably which encodes a polypeptide or protein, and preferably which contains regulatory, non-coding nucleotide sequence(s) present at the 3′ and/or 5′ end of each gene segment. In a preferred embodiment, the gene segment is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO:1-14, inclusive, as described in Table 1, below.

TABLE 1 SEQ ID NO: 1-14, Nucleic Acids of the Invention SEQ ID NO: Description 1 Gene 1 (RRV) - VP1 2 Gene 2 (RRV) - VP2 3 Gene 3 (RRV) - VP3 4 Gene 4 (RRV) - VP4 5 Gene 5 (RRV) - NSP1 6 Gene 6 (RRV) - VP6 7 Gene 7 (RRV) - NSP3 8 Gene 8 (RRV) - NSP2 9 Gene 9 (RRV) (Serotype 3) - VP7 10 Gene 10 (RRV) - NSP4 11 Gene 11 (RRV) - NSP5 12 Gene 9 (DxRRV) (Serotype 1) - VP7 13 Gene 9 (DS1xRRV) (Serotype 2) - VP7 14 Gene 9 (ST3xRRV) (Serotype 4) - VP7

Due to differences in electrophoretic mobility, numerical gene assignments differ among RRV and the reassortant viruses. These differences involve only genes 7, 8 and 9. For the purpose of comparison and discussion, segment 7 is designated as the segment coding for NSP3, segment 8 as the segment coding for NSP2 and segment 9 as the segment coding for outer capsid viral protein 7 (VP7).

The isolated nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA. The RNA or DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense, strand or the non-coding, or antisense, strand. The nucleic acid molecule can include all or a portion of the coding sequence of the gene segment and can further comprise additional non-coding sequences such as non-coding. 3′ and 5′ sequences (including regulatory sequences, for example). Additionally, the nucleic acid molecule can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the protein. Such sequences include, but are not limited to, those which encode a glutathione-S-transferase (GST) fusion protein and those which encode a hemagglutinin A (HA) polypeptide marker from influenza.

An “isolated” nucleic acid molecule, as used herein, is one that is separated from nucleic acids which normally flank the gene or nucleotide sequence and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu (e.g., the virus) in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.

Preferably, an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.

The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant nucleic acid contained in a vector is included in the definition of “isolated” as used herein. Also, isolated nucleic acid molecules include recombinant nucleic acid molecules in heterologous host cells, as well as partially or substantially purified nucleic acid molecules in solution. “Isolated” nucleic acid molecules also encompass in vivo and in vitro RNA transcripts (e.g., of cDNA) of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence which is synthesized chemically or by recombinant means. Therefore, recombinant nucleic acids contained in a vector are included in the definition of “isolated” as used herein. Also, isolated nucleotide sequences include recombinant nucleic acid molecules in heterologous organisms, as well as partially or substantially purified nucleic acid molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules (e.g., cDNA) of the present invention are also encompassed by “isolated” nucleotide sequences. Such isolated nucleotide sequences are useful in the manufacture of the encoded protein, to raise anti-protein antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses, or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis, indicating the presence of infection.

The present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a protein described herein. Thus, for example, nucleic acid molecules which comprise a sequence that is different from the naturally-occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode a protein that is the same as a protein encoded by a gene segment of the present invention, are also the subject of this invention (e.g., a nucleic acid molecule that encodes a protein having as a sequence any one of SEQ ID NO:15-28, as described in Table 2, below).

TABLE 2 SEQ ID NO: 15-28, Proteins Encoded by Nucleic Acids of the Invention SEQ ID Illustrative Nucleic Acid NO: Description Encoding the Protein 15 Protein 1 (RRV) - VP1 1 16 Protein 2 (RRV) - VP2 2 17 Protein 3 (RRV) - VP3 3 18 Protein 4 (RRV) - VP4 4 19 Protein 5 (RRV) - NSP1 5 20 Protein 6 (RRV) - VP6 6 21 Protein 7 (RRV) - NSP3 7 22 Protein 8 (RRV) - NSP2 8 23 Protein 9 (RRV) (Serotype 3) - 9 VP7 24 Protein 10 (RRV) - NSP4 10 25 Protein 11 (RRV) - NSP5 11 26 Protein 9 (DxRRV) (Serotype 1) - 12 VP7 27 Protein 9 (DS1xRRV) (Serotype 13 2) - VP7 28 Protein 9 (ST3xRRV) (Serotype 14 4) - VP7

The invention also encompasses variants of certain nucleotide sequences of the invention. For example, in one embodiment, the variant nucleotide sequences of the invention comprise the nucleotide differences set forth in Table 4 or Table 8, below.

That is, representative valiant embodiments include: a nucleic acid molecule having the sequence of SEQ ID NO: 1 (gene 1) and having a nucleotide change to a G at nucleotide 2120; a nucleic acid molecule having the sequence of SEQ ID NO: 2 (gene 2) and having one or more of the following nucleotide changes: a C at nucleotide 493, and a T at nucleotide 947; a nucleic acid molecule having the sequence of SEQ ID NO:3 (gene 3) and having one or more of the following nucleotide changes: an A at nucleotide 169, a C at nucleotide 283, a C at nucleotide 448, a C at nucleotide 874, a C at nucleotide 1306, and a C at nucleotide 2388; a nucleic acid molecule having the sequence of SEQ ID NO:4 (gene 4) and having one or more of the following nucleotide changes: a C at nucleotide 119, a G at nucleotide 417, a G at nucleotide 809, a C at nucleotide 977, an A at nucleotide 1463, a C at nucleotide 1481, a C at nucleotide 1608, a C at nucleotide 1755, and an A at nucleotide 1953; a nucleic acid molecule having the sequence of SEQ ID NO:5 (gene 5) and having one or more of the following nucleotide changes: an A at nucleotide 75, a T at nucleotide 84, a C at nucleotide 347, a T at nucleotide 667, a C at nucleotide 1186, a G at nucleotide 1219, and an A at nucleotide 1204; a nucleic acid molecule having the sequence of SEQ ID NO: 6 (gene 6) and having one or more of the following nucleotide changes: a C at nucleotide 376, an A at nucleotide 756, an A at nucleotide 1008, and a G at nucleotide 1041; a nucleic acid molecule having the sequence of SEQ ID NO: 7 (gene 7) and having a nucleotide change to a G at nucleotide 387; a nucleic acid molecule having the sequence of SEQ ID NO:10 (gene 10) and having one or more of the following nucleotide changes: an A at nucleotide 92, an A at nucleotide 174, and a G at nucleotide 218; a nucleic acid molecule having the sequence of SEQ ID NO: 11 (gene 11) and having a nucleotide change to A at nucleotide 180; a nucleic acid molecule having the sequence of SEQ ID NO: 12 (DxRRV (serotype 1)) and having a nucleotide change to an A at nucleotide 556; and a nucleic acid molecule having the sequence of SEQ ID NO: 14 (ST3xRRV (serotype 4)) and having a nucleotide change to a G at nucleotide 263. Each gene variant can have one of the nucleotide changes, or can have more than one, or all, of the nucleotide changes enumerated for that particular gene.

Such variants can be naturally-occurring, such as in the case of allelic variation or base substitution in a clinical isolate, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Other intended variations can also be included in any one of the isolated nucleic acids of the invention (e.g., in any one of SEQ ID NO: 1-14 or in a nucleic acid molecule encoding a polypeptide of any one of SEQ ID NO:15-28); such intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides which can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably the nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of protein encoded by the gene segment of the invention.

Other alterations of the nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to designated sequences via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

In a related aspect, the nucleic acid molecules of the invention are used as probes or primers in assays. “Probes” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. Such probes include polypeptide nucleic acids, as described in Nielsen et al., Science, 254, 1497-1500 (1991). Typically, a probe comprises a region of nucleotide sequence that hybridizes under highly stringent conditions to at least about 15, typically about 20-25, and more typically about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID NO: 1-14, and the complement of SEQ ID NO: 1-14. More typically, the probe further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.

As used herein, the term “primer” refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis using well-known methods (e.g., PCR, LCR) including, but not limited to those described herein. The appropriate length of the primer depends on the particular use, but typically ranges from about 15 to 30 nucleotides.

The nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided in SEQ ID NO: 1-14. For example, nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences provided in SEQ ID NO: 1-14 and/or the complement of SEQ ID NO: 1-14. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. The nucleic acid molecules can be amplified using cDNA, RNA, or mRNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.

Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.

Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of SEQ ID NO: 1-14 and/or the complement of SEQ ID NO: 1-14, or a portion of the nucleotide sequence of SEQ ID NO: 1-14 and/or the complements thereof, and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid molecule (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides call be used. Alternatively, the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).

Another aspect of the invention pertains to nucleic acid constructs containing a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1-14 and the complement of SEQ ID NO: 1-14 (or a portion thereof). The constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted hi a sense or antisense orientation. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses, negative strand RNA virus vectors, VEE vectors) that serve equivalent functions.

Preferred recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of protein desired. The expression vectors of the invention can be introduced into host cells to thereby produce proteins, including fusion proteins, encoded by nucleic acid molecules as described herein.

The recombinant expression vectors of the invention can be designed for expression of a protein described herein, in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in Vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a protein described herein. Accordingly, the invention further provides methods for producing a protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a protein described herein has been introduced) in a suitable medium such that the protein is produced. In another embodiment, the method further comprises isolating the protein from the medium or the host cell.

The invention will be further described by the following non-limiting examples. The teachings of all publications cited herein are incorporated herein by reference in their entirety.

EXAMPLES Example 1 Consensus Sequencing Materials and Methods

Viral Stocks

A rotavirus seed bank system was developed for the ROTAMUNE™ vaccine consisting of a Master Virus Seed (MVS) Bank, a Primary Virus Seed (PVS) Bank and a Manufacturer's Working Virus Seed (MWVS) Bank. The nucleotide sequence of all 11 gene segments of each of the four strains comprising the commercial virus seed (MWVS-5, 6, 7 and 8) were identified and the sequence identity (genetic equivalence) between the commercial virus seeds and the clinical virus seeds (MWVS-1, 2, 3, and 4) were demonstrated.

A subset of Manufacturer's Working Virus Seed (WVS), representing clinical lots (MWVS 1-4) and commercial lots (MWVS 5-8), was used. The strains used in this study are:

1) D × RRV Lot MWVS-5 Type-1 2) DS1 × RRV Lot MWVS-6 Type-2 3) RRV Lot MWVS-7 Type-3 4) ST3 × RRV Lot MWVS-8 Type-4 5) RRV Lot MWVS-1 6) DS1 × RRV Lot MWVS-2 7) D × RRV Lot MWVS-3 8) ST3 × RRV Lot MWVS-4

RNA Isolation

Genomic RNAs from the aliquots of each MWVS were extracted using Trizol-LS™ reagent (Life Technologies, Grand Island, N.Y.). RNA was resuspended in RNase-free water and used for all genomic amplifications.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification RT-PCR amplifications spanning individual full length RNA gene segments of the rotavirus genome were performed by using the GeneAmp XL RNA PCR Kit (Perkin-Elmer) and primer pairs specific to each individual rotavirus gene segment. Common RRV primers were used for all segments of the three reassortants, except for gene 9 where primers specific to gene 9 of the rhesus or of the different human rotavirus sequences (RRV, HRV-D, HRV-DS1 and GRV-ST3) were used. Primers are shown in Table 3A.

TABLE 3A Primers Used For Amplification of Genomic Fragments SEQ Gene Primer Name Nucleotide Sequence 5′ → 3′ ID 1 RRV-101  1-27 GGCTATTAAAGCTGTACAATGGGGAAG 29 RRV-102 3265-3294 CTAAGCGTTCTAATCTTGAAAGAAGTTTGC 30 2 RRV-201  1-27 GGCTATTAAAGGCTCAATGGCGTACAG 31 RRV-202 2683-2708 GGTCATATCTCCACAATGGGGTTGGC 32 3 *RRV-301A  1-25 GGCTATTAAAGCAGTACGAGTAGTG 33 *RRV-302A 2571-2591 GGTCACATCATGACTAGTGTG 34 4 RRV-401  1-21 GGCTATAAAATGGCTTCGCTC 35 RRV-402 2341-2362 GGTCACATCCTCTGGAAATTGC 36 5 *RRV-501A  1-29 GGCTTTTTTTTGAAATGTCTTGTGTTAGC 37 *RRV-502A 1576-1599 GGTCACAGTTTTTGCTGGCTAGGC 38 6 RRV-601  1-26 GGCTTTTAAACGAAGTCTTCAACATG 39 RRV-602 1332-1356 GGTCACATCCTCTCACTATACCATC 40 7 RRV-701  1-25 GGCATTTAATGCTTTTCAGTGGTTG 41 RRV-702 1056-1077 GGTCACATAACGCCCCTATAGC 42 8 RRV-801  1-29 GGCTTTTAAAGCGTCTCAGTCGCCGTTCG 43 RRV-802 1037-1059 GGTCACATAAGCGCTTTCTATTC 44 9 **RRV-901  1-24 GGCTTTAAAAGCGAGAATTTCCGT 45 RRV-902 1041-1062 GGTCACATCATACATTTCTAAC 46 D-RRV-902 1036-1062 GGTCACATCGAACAATTCTAATCTAAG 47 DS1-RRV-902 1035-1062 GGTCACATCGAACAATTCTGACCAAATC 48 ST3-RRV-902 1036-1062 GGTCACATCATACATTTCTATTTTAGG 49 10 RRV-1001  1-27 GGCTTTTTAAAAGTTCTGTTCCGAGAG 50 RRV-1002 728-750 GGTCACATTAAGACCGTTCCTTC 51 11 RRV-1101  1-24 GGCTTTTAAAGCGCTACAGTGATG 52 RRV-1102 644-667 GGTGACATAACTGGAGTGGGGAGC 53 Primers named . . . 01 are (+) sense and primers named . . . 02 are (−) sense. *Primers 301A, 302A, 501A, and 502A reflect the actual termini sequence as determined. **Primer 901 was used to amp1ify all four serotypes, while primer 902 was designed specifically for each.

RT-PCR amplification steps were as follows:

RT Reaction

RNA (RNA + water)  10 μl RNase-free water — 5X XL RT buffer   4 μl dNTP mix (2.5 mM each dNTP) 1.6 μl rTth polymerase 2.5 μl 25 mM Mn(OAc2) 0.88 μl  RT (downstream) primer (20 pmoles/μl)   1 μl TOTAL:  20 μl

RNA (100-500 ng per reaction) was mixed with water and 5×SL RT buffer in a Gene-amp tube. The mixture was denatured at 96° C. for 5-6 minutes in a pre-heated thermal cycler, and then placed on ice immediately for 3 minutes to quick cool. Samples were pulse-spun in microfuge to remove any condensation on caps, and the remaining ingredients were added. A drop of mineral oil was added to each tube, and samples were placed in pre-heated thermal cycler at 40° C. The RT thermal cycle was 40° C. for 60 minutes, followed by 45° C. for 60 minutes and then 4° C., soak. If several different fragments from the same RNA template were amplified, the entire reaction was scaled up in one tube, eliminating only the primer from the mix. One μl of the RT (+sense) primer was added to each reaction tube, and then 19 μl of the scaled-up RT mix were added.

PCR Reaction

RNase-free water 58.8 μl  5X XL Chelating Buffer  16 μl 25 mM Mg(OAc2) 3.2 μl PCR (upstream) primer (20 pmoles/μl) 1.5 μl RT (downstream) primer (20 pmoles/μl) 0.5 μl TOTAL  80 μl.

The reagents were mixed for each sample; 80 μl PCR mix were added to the 20 μl RT reaction under the oil, and mixed by pipetting up and down several times. The samples were placed in pre-heated thermal cycler (hot-start) at 94° C. and cycled as follows: 94° C. denaturation for 3 minutes, followed by 40 cycles of denaturation at 94° C. for 1 minute, primer annealing at 40° C. for 30 seconds, and extension at 70° C. for five minutes. A final extension step was run at 70° C. for 10 minutes, followed by a soak cycle at 4° C.

Ten μl of reaction were run on agarose gel. Products were purified using Promega's Wizard PCR preps DNA purification system (Madison Wis.) either directly or using gel purification from low-melting agarose.

RNA Ligation RT-PCR to Determine Consensus Nucleotide sequence of the Gene Termini

The nucleotide sequence of the absolute 3′ and 5′ termini of the eleven RNA gene segments of the rotavirus vaccine strains was determined using an RNA ligation reverse transcription-PCR protocol modified from Sidhu et al. (Virology 193:66-72, 1993) for use on double-stranded RNA. Genomic RNA was extracted using Trizol-LS™ reagent (Life Technologies) and 2-3 μg was treated with Tobacco Acid Pyrophosphatase (TAP) at 37° C. for 1 hour in a 40 μl volume according to the manufacturer's directions (Epicentre Technologies). This step was used to remove the CAP structure on the 5′ end of the plus strand of the genomic RNA segments, and leave behind a 5′ monophosphate that was used for RNA ligation. The TAP-treated RNA was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol-precipitated. The RNA pellet was air-dried and resuspended in 13.7 μL of RNase-free water. The double-stranded RNA was then denatured at 96° C. for 4 minutes and quick-chilled on ice for 2 minutes. The reagents required for RNA ligation in at 20 μl volume were then added. The final ligation reaction conditions were 1×RNA ligase buffer (New England Biolabs), 10% DMSO (Sigma D-2650), 20 U Promega RNasin, and 36 U NEB T4 RNA ligase. Ligation was performed at 16° C. overnight (approximately 16 hours). The ligated RNA was phenol extracted and ethanol precipitated as described above, and the RNA pellet was resuspended in 15 μl of water. One μl of ligated RNA was seeded into each of 11 RT-PCR reactions containing rotavirus gene-specific primers (shown in Table 3B) designed to amplify across the ligated RNA junction.

TABLE 3B Rotavirus Primers Used to Amplify across Ligated RNA Junctions (Gene Termini) Gene Primer Nucleotide Sequence 5′ → 3′ SEQ ID 1 RRV-101T 206-229 CCCAAGCTTGTGTGGCATTCTCTATAACATCGC 54 RRV-102T 3163-3186 CCCGGATCCGGCTCATGGATAAGCTTATTCTGC 55 RRV-103T 301-324 CCCAAGCTTCAAATCTGCTTCTAGCGGCTTACC 56 RRV-104T 3084-3107 CCCGGATCCTAAAGGAAAGATACCAGCTGTCAC 57 2 RRV-201T 147-170 CCCAAGCTTCCTTTTGAGATAGCACTTTCTCTG 58 RRV-202T 2524-2547 CCCGGATCCACCACAACAATTTGATTTTAGAGC 59 RRV-203T 270-293 CTTCTTTTTGATGTTCTTCCTTAG 60 RRV-204T 2470-2493 TGTAGCGAATTATGACTGGGTTCG 61 3 RRV-301T 128-151 CCCAAGCTTAAGAAATGCATTCTCGTAACTGTC 62 RRV-302T 2415-2438 CCCGGATCCGTCAATCTAGAATGTTTATTCCAC 63 RRV-303T 227-250 TTGAATCTCAACAGCTGCAATTCC 64 RRV-304T 2344-2367 AAACGTTAGTGGAGTTCTAGCGAC 65 4 RRV-401T 137-160 CCCAAGCTTCCCAGTTAACTGGAGCATAACCTG 66 RRV-402T 2145-2168 CCCGGATCCAACTGACTCTCCGGTCATCTCAGC 67 RRV-403T 208-231 GAACGTTGTTGGTTGATAAGGACC 68 RRV-404T 2049-2072 AGTCTTTGAAGCGGGAACAGATGG 69 5 RRV-501T 155-178 CCCAAGCTTATTGACAACACTCAATGCACCACC 70 RRV-502T 1396-1419 CCCGGATCCGAATTAGATCACTTGCCGTTATGC 71 RRV-503T 214-237 GATGCACCACTGACAAACATGAGC 72 RRV-504T 1302-1325 GATACTGGAAACCGAGGCTCTTCC 73 6 RRV-601T 166-189 CCCAAGCTTTCGGCAGATTACCAATTCCTCCAG 74 RRV-602T 1170-1193 CCCGGATCCCAGCGTGTATTTACAGTGGCTTCC 75 RRV-603T 246-269 ACGGGCCGTTTCGACATAGTTAGC 76 RRV-604T 1076-1099 AGAATACGCGATACCAGTTGGACC 77 7 RRV-705T  94-117 CCCAAGCTTTCAAGAGTAGAAGTTGCAGCAACC 78 RRV-706T 923-946 CCCGGATCCAAAGGATTATTGCAGCAATGCAAC 79 RRV-703T 146-169 ACTCTTTACTCTAGTATATACCTC 80 RRV-704T 800-823 AAATCAGACATTGAACAACAGCTG 81 RRV-707T 285-311 TAGCTACAGTTCGAGAGTCAGTCATCC 82 RRV-708T 756-782 CAATAGAATGGTATCTAAGATCGATGG 83 8 RRV-801T 194-217 CCCAAGCTTGAATTGTGGCGGTGGTGCGATACC 84 RRV-802T 920-943 CCCGGATCCAAAATGAAGCGGGAAAGTAATCCG 85 RRV-803T 284-307 CGCTTCACAAATTAACACCGCCAC 86 RRV-804T 847-870 GAACTGGTATGCGTTTACATCCTC 87 9 (RRV) RRV-901T 227-250 CCCAAGCTTCGTATGCAGTGTCCATTGAACCAG 88 RRV-902T 905-928 CCCGGATCCGCATTAATTGGAAGAAATGGTGGC 89 RRV-903T 304-327 TATTTCTGTTGCAGCTTCAGTTGG 90 RRV-904T 835-858 GTTGGAGGTTCTGATGTTCTCGAC 91 9 (DSI) DS1-901T 134-157 CCCAAGCTTACCTGAAAATTATGTAGTCCATCG 92 DS1-902T 882-905 CCCGGATCCCCCACAAGTTCAAAGAATCATGCG 93 DS1-903T 220-243 AGCGTCTAGTGACCCCGTTATTGG 94 DS1-904T 828-851 AATTCAAGTTGGTGGACCGAACGC 95 9 (D) D-901T 122-145 CCCAAGCTTTGTAGTCCATTATTCGAGTCACTG 96 D-902T 886-909 CCCGGATCCCAAACTGAGAGAATGATGAGAGTG 97 D-903T 220-243 AGCGTCCATTGATCCTGTTATTGG 98 D-904T 819-842 TGTAGCTGTAATACAAGTTGGTGG 99 9 (ST3) ST3-901T 219-242 CCCAAGCTTGTATCCATAGATCCAGTAATTGGC 100 ST3-902T 920-943 CCCGGATCCAATGGTGGCAAGTATTCTACACTG 101 ST3-903T 304-327 AATTTGAGTTGGAGCTTCTGATGG 102 ST3-904T 861-884 AACAGCTGATCCCACAACTTCTCC 103 10 RRV-1005T 141-168 GTACAGTTAGGACAGAAGCAATGTATGG 104 RRV-1006T 628-655 ATCGGACCTGATGACTGGTTGAGAAGCC 105 RRV-1007T 359-386 CTTATCAATCATTTCCAGCTGACGTCTC 184 RRV-1008T 533-559 TCCATATGAACCAAAAGAGGTGACTGC 185 11 RRV-1101T 124-147 CCCAAGCTTTGAAACGTACTGTTCACTCCTACC 106 RRV-1102T 470-493 CCCGGATCCTTGAAGCAGATTCCGATTCAGACG 107 RRV-1103T 205-228 CCCAAGCTTGTCGTTTGAAGCAGAATCAGATGG 108 RRV-1104T 403-426 CCCGGATCCGTATCAACAGTTTCCAAGAAGGAG 109 Odd numbered primers are negative sense; Even numbered primers are positive sense. Primers ending with 03, 04 or 07, 08 (genes 7 and 10) are used for reverse transcription and first round PCR. Primers ending with 01, 02 or 05, 06 (genes 7 and 10) are used for second round (nested) PCR and sequencing. Primers ending with 01 and 103T, 1103T, 705T contain 9 nucleotides at the 5′ end that include a HindIII site. Primers ending with 02 and 104T, 1104T, 706T contain 9 nucleotides at the 5′ end that include a BamHI site. The RT step and the first round of PCR was done using the Perkin-Elmer GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (catalog #N808-0069) as per the manufacturer's specifications with modification.

RT mix for one reaction (multiply μl volumes by number of reactions needed)

10X RT buffer 2 2.5 mM dNTP mix* 1.6 10 mM MnCl2 2 rTth DNA polymerase (2.5 U/μl) 2 RNase-free water 7.4 TOTAL: 15 μl *prepared by mixing equal volumes of each 10 mM dNTP stock (dATP, dGTP, dCTP, dTTP). Fifteen μl of RT mix were combined with 2 μl of upstream and 2 μl of downstream first round PCR primers (each are 20 pmoles/μl) and 1 μl of ligated RNA in a 0.5 ml thin-walled GeneAmp tube and overlaid with 2 drops of Sigma mineral oil. Since both plus and minus RNA strands of the genome have been ligated, each of the first round PCR primers could be used for cDNA synthesis during reverse transcription. For each different primer pair used, a negative control was set up that contained 1 μl of water in place of the ligated RNA. The 0.5 ml reaction tubes were loaded in the PE thermal cycler 480 at 4° C. and the cycler was quickly ramped to 80° C. and then ramped back to 45° C. RT was performed at 45° C. for 30 minutes, followed by 50° C. for 30 minutes. Following RT, 80 μl of first-round PCR mix was added to each RT reaction over the oil and the tubes were pulse spun in a microcentrifuge.

First Round PCR Mix for one reaction (multiply III volumes by the number of reactions needed):

2.5 mM dNTP mix 2.4 25 mM MgCl2 9 10x chelating buffer 8 RNase-free water 60.0 TOTAL 80 μl Thermal cycling profile was as follows: 94° C. for 2 minutes; 40 cycles of 94° C. for 1 minute, 45° C. for 1 minute, 72° C. for 1 minute; 72° C., for 10 minutes; and 4° C., soak. Following each first round PCR, a second round (nested) PCR amplification was performed using Perkin-Elmer reagents and AmpliTaq Gold™ DNA polymerase (catalog #N808-0241).

Second Round (nested) PCR mix for one reaction (multiply μl volumes by the number of reactions needed)

10x PCR buffer II (catalog #N808-0010) 10 25 mM MgCl2 8 2.5 mM dNTP mix 8 AmpliTaq Gold DNA polymerase (5 U/μl) 0.5 RNase-free water 65.5 TOTAL: 92 μl The second round PCR mix (92 μl) was combined with 2 μl of upstream and 2 μl of downstream second round PCR primers (each are 20 pmoles/μl) and 4 μl of the first round PCR reaction (including negative controls). The reactions were overlaid with 2 drops of Sigma mineral oil and pulse spun. The thermal cycling profile was the same as for the first round PCR except the initial step at 94° C. for 2 minutes was extended to 12 minutes to activate the AmpliTaq Gold™ DNAP.

Second round PCR products (10 μl) were analyzed by agarose gel electrophoresis with ethidium bromide. The ligation PCR products were gel-purified using the Promega Wizard™ PCR preps DNA purification system. A consensus sequence for the PCR amplified products was determined. If necessary to resolve nucleotide sequence ambiguities, the PCR products were cloned using pGEM-T Easy Vector System I Promega, Madison, Wis.) and multiple clones were sequenced.

DNA Sequencing

A consensus sequence for the PCR amplified products was generated by using the Applied Biosystems-PRISM fluorescent dye terminator cycle sequencing kit with AmpliTaq DNA Polymerase-FS, and the Applied Biosystems 377 DNA sequencer (ABI-Perkin-Elmer). Over 100 primers spaced approximately 200 nucleotides apart on each strand were used for sequencing both strands of the PCR products. When needed, gel purified PCR products were cloned by using pGEM-T Easy Vector System I (Promega, Madison, Wis.). Positive clones were selected by T7/SP6 primer-specific PCR screening and the amplified PCR products of the positive clones were directly sequenced as described above. Sequences were analyzed by using MacVector gene analysis program (Oxford Molecular, Oxford, UK).

Results

Eleven full length gene segments were amplified for each virus strain using high fidelity RNA-PCR amplification reactions as described above. Both strands of the amplified products were sequenced directly by using RRV-specific primers, except for gene segment 9, for which strain specific primers were used. The sequences for each of the eleven RRV (MWVS-7) genes are SEQ ID NO: 1-11, respectively as shown in Table 1 above. Gene segment 9 sequences for the three reassortants are SEQ ID NO:12 (DxRRV (MWVS-5)), SEQ ID NO:13 (DS1xRRV (MWVS-6)), and SEQ ID NO: 14 (ST3xRRV (MWVS-8)). The putative protein sequences for each of these are SEQ ID NO: 15-28, respectively, as shown in Table 2 above.

Table 4 lists nucleotide differences identified between the parent RRV (MWVS-7) strain and the three reassortant viruses D×RRV (MWVS-5), DS1×RRV (MWVS-6), and ST3×RRV (MWVS-8). These nucleotide differences were common to both clinical (MWVS 1-4) and commercial (MWVS 5-8) seeds.

TABLE 4 List of nucleotide changes identified in the gene segments of the three reassortants as compared to the progenitor RRV strain MWVS-5 MWVS-6 & MWVS-8 & MWVS- MWVS-2 & MWVS- Nucleotide 3 (D × (DS1 × 4 (ST3 × Amino Gene Position RRV) RRV) RRV) Acid 1 2120  A→G — — Lys→Arg 2  493* — — A→C silent 947 C→T — — Leu→Phe 4 119 T→C T→C — Leu→Pro 809 A→G A→G — Tyr→Cys 977 T→C — — Met→Thr 1463  — — C→A Thr→Asn 1755  T→C — — silent 5  84 C→T — — silent 347 — T→C — Leu→Ser 6 376 — — A→C Lys→Thr 756 G→A G→A G→A Ala→Thr 1008  G→A — — Ala→Thr 1041  A→G — — Lys→Glu 7 387 — A→G A→G Asn→Ser 10 174 G/A mix† — G/A mix† Ala/Thr mix 218 A→G — — silent 11 180 G→A — — silent — Identical to RRV. †Position 174 is an ‘A’ in RRV (MWVS-1 and MWVS-7).

Example 2 Clonal Analysis

Materials and Methods

Viral Stocks

Viral stocks were from the Wyeth Laboratories, Inc., in Marietta, Pa., USA, rotavirus seed bank system. The strains used in this study are:

Working virus seeds for commercial vaccine production:

1) D × RRV Lot MWVS-5 Type-1, Serotype G1 2) DS1 × RRV Lot MWVS-6 Type-2, Serotype G1 3) RRV Lot MWVS-7 Type-3, Serotype G3 4) ST3 × RRV Lot MWVS-8 Type-4, Serotype G4 Working virus seeds for clinical vaccine production:

5) RRV Lot MWVS-1 (ST3), Serotype G3 6) DS1 × RRV Lot MWVS-2 (TS2), Serotype G2 7) D × RRV Lot MWVS-3 (ST1), Serotype G1 8) ST3 × RRV Lot MWVS-4 (ST4), Serotype G4 Commercial vaccine lots used in clonal analysis were as follows:

Serotype G1: I973020, I973017, I983003, I983026 Serotype G2: I973030, I973029, I983008, I983037 Serotype G3: I973026, I983021, I983032 Serotype G4: I97003, I973040, I983030, I973034.

All clinical isolates were plaque purified three times, except for isolates 37 and 38 (purified twice).

Serotype G1 clinical isolates: clones 1, 16, 22 and 29

Serotype G2 clinical isolates: clones 3, 4, 8, 9 and 19

Serotype G3 clinical isolates: clones 5, 6, 10, 11, 12, 20, 21 and 25

Serotype G4 clinical isolates: clones 37 and 38.

RNA Isolation

Total RNA was extracted from clinical samples, aliquots of virus seed or aliquots of vaccine virus using Trizol-LS™ reagent (Life Technologies, Grand Island, N.Y.). RNA was resuspended in nuclease-free water and used for all RT/PCR amplifications.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Amplification

Clonal analysis was carried out to determine the following: 1) the micro-heterogeneity in MWVS-1, 2, 3 and 4 at the 7 nucleotide positions (position 2388 in gene 3, positions 1674 and 1953 in gene 4, positions 75, 667 and 1204 in gene 5, and position 556 in gene 9) which were found to differ from consensus sequence in more than one clinical isolate; 2) the micro-heterogeneity in the four seed strains (MWVS-1, 2, 3 and 4) at position 174 in gene 10, found to contain a base mixture in serotypes G1 and G4 by consensus sequencing; and 3) the micro-heterogeneity in genes 3 and 10 at nucleotide positions 2388 and 174, respectively, in the commercial vaccine lots and their seeds (MWVS-5, 6, 7 and 8).

A portion of each gene spanning the nucleotide position(s) of interest was amplified by RT-PCR using the GeneAmp XL RNA PCR Kit (Perkin-Elmer) and primer pairs listed in Table 5.

TABLE 5 Primer Pairs Used in RT/PCR Amplification of Clinical and Commercial Seeds for Clonal Analysis Nucleotide Primer Primer Sequence SEQ Gene Of Interest Name (5′ -> 3′) ID 3 2388 RRV-307 GCTGATATAGAAGGTGGAAAG 110 RRV-302A GGTCACATCATGACTAGTGTG 111 4 1674 RRV-406 GAGCACCATAGATGCAGCT 112 RRV-409 CTGACAGATGAAGAAACATC 113 1953 RRV-406 GAGCACCATAGATGCAGCT 114 RRV-408 GAGTCAGTTACTAGATCTGC 115 5 75, 667, RRV-501A GGCTTTTTTTTGAAATGTCT 116 1204 TGTGTTAGC RRV-507 GTGCATAACGGCAAGTGATC 117 9 (G1) 556 RRV-901 GGCTTTAAAAGCGAGAATTT 118 CCGT DxRRV- GGTCACATCGAACAATTCTA 119 902 ATCTAAG 10 174 RRV-1001 GGCTTTTAAAAGTTCTGTTC 120 CGAGAG RRV-1002 GGTCACATTAAGACCGTTCC 121 TTC Primers common to all four serotypes were used for RT-PCR amplification of fragments from all genes except gene 9 where primers specific to gene 9 of human rotavirus serotype G1 (HRV-D) were used. RT-PCR amplification was carried out as follows:

RT Reaction

RNA (RNA + water)  10 μl RNase-free water — 5X XL RT buffer   4 μl dNTP mix (2.5 mM each dNTP) 1.6 μl rTth polymerase 2.5 μl 25 mM Mn (OAc2) 0.88 μl  RT (downstream) primer (20 pmoles/μl)   1 μl TOTAL:  20 μl RNA (100-500 ng per reaction) was mixed with water and 5× SL RT buffer in a Gene-amp tube. The mixture was denatured at 96° C. for 4 minutes in a preheated thermal cycler, and then placed on ice immediately for 3 minutes to quick cool. Samples were pulse-spun in microfuge to remove any condensation on caps, and the remaining ingredients were added. A drop of mineral oil was added to each tube, and samples were placed in pre-heated thermal cycler at 40° C. The RT thermal cycle was 40° C. for 60 minutes, followed by 45° C. for 60 minutes and then 4° C., soak. If several different fragments from the same RNA template were amplified, the entire reaction was scaled up in one tube, eliminating only the primer from the mix. One μl of the RT (+sense) primer was added to each reaction tube, and then 19 μl of the scaled-up RT mix were added.

PCR Reaction

RNase-free water 58.8 μl  5X XL Chelating Buffer  16 μl 25 mM Mg (OAc2) 3.2 μl PCR (upstream) primer (20 pmoles/μl) 1.5 μl RT (downstream) primer (20 pmoles/μl) 0.5 μl TOTAL  80 μl. The reagents were mixed for each sample; 80 μl PCR mix were added to the 20 μl RT reaction under the oil, and mixed by pipetting up and down several times. The samples were placed in pre-heated thermal cycler (hot-start) at 94° C. and cycled as follows: 94° C. denaturation for 3 minutes, followed by 40 cycles of denaturation at 94° C. for 1 minutes primer annealing at 40° C. for 30 seconds, and extension at 70° C. for two minutes. A final extension step was run at 70° C. for 10 minutes, followed by a soak cycle at 4° C.

Ten μl of reaction were run on 1% agarose gel. Products were purified using Promega's Wizard PCR preps DNA purification system (Madison Wis.) either directly or using gel purification from low-melting agarose.

Cloning of RT-PCR Products

The RT-PCR products from genes 3, 4, 5, 9 and 10 of MWVS-1, 2, 3 and 4 were cloned into the appropriate restriction endonuclease sites of either pGEM-3Zf(+)™, pGEM-5Zf(+)™, or pGEM T-Easy™ (T/A cloning) plasmid vectors using standard cloning methods.

Screening for Positive Clones

Approximately 40-100 colonies from each plasmid construct were screened for the presence of cloned RRV sequences by PCR using primers specific to the SP6 and T7 promoter sequences (SP6:5′TATTTAGGTGACACTATAG3′) (SEQ ID NO.: 122) (T7:5′TAATACGACTCACTATAGGG3′) (SEQ ID NO.: 123) flanking the cloning sites of each vector as follows: a single colony was transferred to 10 μl of water in a 0.5 ml Gene-Amp® tube using a sterile inoculating needle, and overlaid with a drop of mineral oil. The tubes were placed in a pre-heated thermal cycler at 96° C. for 10 minutes to lyse bacterial cells, and cooled to 4° C. for 3-4 minutes. Then, 40 μl PCR mix was added to each tube underneath the oil and pipetted several times to mix the sample.

PCR MIX (50 reactions):

10X PCR buffer with MgCl₂ (Perkin-Elmer) 200 μl  dNTP mix (2.5 mM each dNTP) (Perkin-Elmer) 250 μl  SP6 promoter primer (20 pmole/μl) 50 μl T7 promoter primer (20 pmole/μl) 50 μl Nuclease-free water 1438 μl  Taq DNA polymerase (Perkin-Elmer) 5 U/μl 12 μl TOTAL 2000 μl  The mixture was placed in a pre-heated thermal cycler (hot-start) at 94° C. and cycled as follows: initial 94° C. denaturation for 3 minutes, followed by 40 cycles of denaturation at 94° C. for 1 minute, primer annealing at 42° C. for 1 minute, and extension at 72° C. for 2 minutes, terminating with a soak file at 4° C. Each PCR product (10 μl) was run on a 1% agarose gel to confirm the presence of an insert of the appropriate size.

Sequence Determination of Positive Clones

PCR products from 25-30 positive clones were directly purified from the PCR reaction using the PCR Preps Kit (Promega) and eluted in 25 μl nuclease-free water. Three μl of the purified product were then sequenced using Taq Cycle Sequencing terminator mix, FS (ABI) and primers specific for the SP6 and T7 promoters which flank the MCS of each plasmid vector.

Sequence Configuration of Clinical Virus Isolates

RNA was extracted from 400 μl of each clinical virus isolate as previously described. For each virus, a fragment containing the nucleotide position(s) of interest was amplified from 200-600 ng of RNA using the GeneAmp XL RNA PCR Kit (Perkin-Elmer) and primer pairs shown in Table 6. Consensus sequence of the resulting RT/PCR product, including the nucleotide of interest, was determined using Taq cycle sequencing and the ABI 377 DNA sequencer with primers denoted in Table 6.

TABLE 6 Primers Used for RT/PCR Amplification and Sequence Confirmation of Clinical Virus Isolates Gene Clone # # (Serotype) NT # Primer Pairs For RT/PCR and Sequencing 3  3 (G2) 283 301A → GGCTATTAAAGCAGTACGAGTAGTGTG SID 313 → CGTGCTATCGGTAAAGAAGTAGT SID  4 (G2) 2288 307 → GCTGATATAGAAGGTGGAAAG 124 302A → GGTCACATCATGACTAGTGTG 125  5 (G3) 1306 305 → GACTGCTATGGATTTAGAGC 126 311 → GATAATGCGTATAATGCCAC 127  6 (G3) 169 301A → GGCTATTAAAGCAGTACGAGTAGTGTG 128 314 → CTCAACAGCTGCAATTCCTG 129  9 (G2) 2388 307 → GCTGATATAGAAGGTGGAAAG 130 302A → GGTCACATCATGACTAGTGTG 131 21 (G3) 2388 307 → GCTGATATAGAAGGTGGAAAG 132 302A → GGTCACATCATGACTAGTGTG 133 25 (G3) 308 301A → GGCTATTAAAGCAGTACGAGTAGTGTG 134 313 → CGTGCTATCGGTAAAGAAGTAGT 135 29 (G1) 874 304 → GTCTCAGTTGGACATTGGAC 136 312 → GAATATGGAGTGTCAAGTGGGTC 137 38 (G4) 448 301A → GGCTATTAAAGCAGTACGAGTAGTGTG 138 313 → CGTGCTATCGGTAAAGAAGTAGT 139 4  1 (G1) 1953 406 → GAGCACCATAGATGCAGCT 140 408 → GAGTCAGTTACTAGATCTGC 141  6 (G3) 1608 405 → CAGTAATGACTGGCGGAGCAGT 142 409 → CTGACAGATGAAGAAACATC 143 11 (G3) 417 401 → GGCTATAAAATGGCTTCGCTC 144 412 → GTCACAAAATGCTGTCATG 145 12 (G3) 1481 405 → CAGTAATGACTGGCGGAGCAGT 146 409 → CTGACAGATGAAGAAACATC 147 16 (G1) 1953 406 → GAGCACCATAGATGCAGCT 148 408 → GAGTCAGTTACTAGATCTGC 149 19 (G2) 1674 405 → CAGTAATGACTGGCGGAGCAGT 150 409 → CTGACAGATGAAGAAACATC 151 20 (G3) 1674 405 → CAGTAATGACTGGCGGAGCAGT 152 402 → GGTCACATCCTCTGGAAATTGG 153 5  6 (G3) 75 501A → GGCTTTTTTTTGAAATGTCTTGTGTTAGC 154 509 → GTCTATATGGCAAATCTATGC 155 20 (G3) 75 501A → GGCTTTTTTTTGAAATGTCTTGTGTTAGC 156 509 → GTCTATATGGCAAATCTATGC 157  3 (G2) 1204 504 → CTCAAACTGATTTACATCATG 158 507 → GTGCATAACGGCAAGTGATC 159  4 (G2) 1204 504 → CTCAAACTGATTTACATCATG 160 507 → GTGCATAACGGCAAGTGATC 161  5 (G3) 394 501A → GGCTTTTTTTTGAAATGTCTTGTGTTAGC 162 509 → GTCTATATGGCAAATCTATGC 163  8 (G2) 667 503 → CAGAGGAAATGTAGAAATGAG 164 508 → CAATCCATGTCTCTGAATGC 165  9 (G2) 667 503 → CAGAGGAAATGTAGAAATGAG 166 508 → CAATCCATGTCTCTGAATGC 167 10 (G3) 1186 504 → CTCAAACTGATTTACATCATG 168 507 → GTGCATAACGGCAAGTGATC 169 29 (G1) 1219 504 → CTCAAACTGATTTACATCATG 170 507 → GTGCATAACGGCAAGTGATC 171 9 22 (G1) 556 901 → GGCTTTAAAAGCGAGAATTTCCGT 172 D-902 → GGTCACATCGAACAATTCTAATCTAAG 173 29 (G1) 556 901 → GGCTTTAAAAGCGAGAATTTCCGT 174 D-902 → GGTCACATCGAACAATTCTAATCTAAG 175 37 (G4) 263 901 → GGGTTTAAAAGCGAGAATTTCCGT 176 ST3-905 → GTACATGATGATCCCATTGA 177 10 22 (G1) 92 1001 → GGCTTTTTAAAAGTTCTGTTCCGAGAG 178 1002 → GGTCACATTAAGACCGTTCCTTC 179 22 (G1) 174 1001 → GGCTTTTTAAAAGTTCTGTTCCGAGAG 180 1002 → GGTCACATTAAGACCGTTCCTTC 181 22 (G1) 218 1001 → GGCTTTTTAAAAGTTCTGTTCCGAGAG 182 1002 → GGTCACATTAAGACCGTTCCTTC 183

Results

Analysis of Micro-Heterogeneity in Clinical Virus Seeds (MWVS-1, 2, 3 and 4)

Sequence analysis of genes 3, 4, 5, 9 and 10 of the 19 clinical viral isolates obtained from the stools of ROTAMUNE™ recipients had heterogeneity at 24 nucleotide positions when compared to consensus sequence of the clinical seeds (MWVS-1, 2, 3 and 4). One of these sites, nucleotide position 174 of gene 10, contains a base mixture in the serotype G1 and G4 viruses. In addition, 7 of the 24 nucleotide base substitutions identified in the clinical virus isolates (i.e., position 2388 in gene 3; positions 1674 and 1953 in gene 4; positions 75, 667 and 1204 in gene 5; and position 556 in gene 9) were observed in more than one virus sample. To establish the precise level of microheterogeneity present at nucleotide position 174 of gene 10, as well as to determine whether the seven nucleotide base substitutions found in more than one clinical virus isolate were the result of sequence micro-heterogeneity in the clinical virus seeds, clonal analysis was carried out on each of the four seeds at these eight nucleotide positions.

The clonal analysis revealed micro-heterogeneity at position 2388 in gene 3 of the G2 clinical seed (MWVS-2), and at position 174 in gene 10 of the G1, G21 and G4 virus seeds (MWVS-3, 2 and 4, respectively). Twenty-six percent of the viral genomes in the G2 clinical seed were found to contain minor species C at position 2388 of gene 3, while the remaining 74% of the genomes contained T at this position. As predicted by consensus sequence, heterogeneity (i.e., G>A) was observed for position 174 of gene 10 in the G1 (MWVS-3) and G4 (MWVS-4) clinical seeds, where the minor species A was found in 12% and 7% of the genomes, respectively. In contrast, the genomes of the RRV parental strain contained solely A at this position. The analysis also revealed a minor population of 5% G at the same position in the G2 virus seed (MWVS-2) which was not detected by consensus sequencing.

The remainder of the nucleotide substitutions which occur in more than one of the clinical isolates (i.e., positions 1674 and 1953 of gene 4; positions 75, 667 and 1204 of gene 5; and position 556 of gene 9) apparently do not result from measurable sequence micro-heterogeneity within the virus seeds, since mixtures of bases were not found at these positions by clonal analysis. Table 7 summarizes the micro-heterogeneity found in the clinical virus seeds (MWVS-1, 2, 3 and 4) at the variable nucleotide positions. The micro-heterogeneity at these positions may exist at levels below the detection limits, or the substitutions observed in the clinical isolates may represent adaption within the human gastrointestinal tract.

Sequence Confirmation of Clinical Virus Isolates

The sequence of the clinical virus isolates at each of the 24 nucleotide positions where the sequence had been found to diverge from consensus was re-examined. Table 8 lists the 24 nucleotide positions of heterogeneity.

Analysis of the Genetic Stability of RRV at Positions 2388 and 174 in Genes 3 and 10, Respectively

Consensus sequencing of the commercial seeds (MWVS-5, 6, 7 and 8) revealed heterogeneity at positions 2388 and 174 in genes 3 and 10, respectively. To compare the precise levels of micro-heterogeneity in the clinical and commercial virus seeds at these positions, as well as to analyze the genetic stability of these nucleotide positions during vaccine manufacture, clonal analysis of these positions was carried out in the commercial virus seeds and several vaccine lots generated from them.

The data showed that a similar level of micro-heterogeneity existed in the commercial (MWVS-5, 6, 7 and 8) and clinical (WVS-1, 2, 3 and 4) vaccine seeds at positions 2388 and 174 in genes 3 and 10, respectively. By clonal analysis, 15% of the minor species (C) was observed at nucleotide position 2388 in the G2 commercial virus seed (MWVS-2), compared to 26% C in the G2 clinical virus seed (MWVS-2), as

Serotype 1-MWVS-3=DxRRV; Serotype 2=MWVS-2=DS1xRRV; Serotype 3=MWVS-1=RRV; Serotype 4=MWVS-4=ST3xRRV

TABLE 7 Micro-Heterogeneity within clinical virus seeds (MWVS 1, 2, 3 and 4) Nucleotide Nucleotide Representing Representing Genomic Consensus Minor Number of Serotype Nucleotide (% Variant (% Clones Gene (Virus Seed) Position population) population) Sequenced 3 1 (MWVS-3) 2388 T (100%) C (0%) 28 2 (MWVS-2) T (74%) C (26%) 27 3 (MWVS-1) T (100%) C (0%) 60 4 (MWVS-4) T (100%) C (0%) 26 4 1 (MWVS-3) 1674 A (100%) G (0%) 26 2 (MWVS-2) A (100%) G (0%) 24 3 (MWVS-1) A (100%) G (0%) 33 4 (MWVS-4) A (100%) G (0%) 31 1 (MWVS-3) 1953 C (100%) A (0%) 28 5 1 (MWVS-3) 75 C (100%) A (0%) 25 2 (MWVS-2) C (100%) A (0%) 20 3 (MWVS-1) C (100%) A (0%) 20 4 (MWVS-4) C (100%) A (0%) 20 1 (MWVS-3) 667 C (100%) T (0%) 26 2 (MWVS-2) C (100%) T (0%) 19 3 (MWVS-1) C (100%) T (0%) 20 4 (MWVS-4) C (100%) T (0%) 20 1 (MWVS-3) 1204 G (100%) A (0%) 26 2 (MWVS-2) G (100%) A (0%) 20 3 (MWVS-1) G (100%) A (0%) 20 4 (MWVS-4) G (100%) A (0%) 21 9 1 (MWVS-3) 556 G (100%) A (0%) 41 10 1 (MWVS-3) 174 *G (88%) *A (12%) 26 2 (MWVS-2) A (95%) G (5%) 22 3 (MWVS-1) A (100%) G (0%) 21 4 (MWVS-4) *G (93%) *A (7%) 28 *Mixed base depicted in MWVS-3 and MWVS-4 by consensus sequencing (see Table 4).

TABLE 8 Nucleotide Sequence Differences Between Clinical Virus Isolates and Consensus Sequence of MWVS. Clone # Gene # (Serotype) NT # NT Change AA # AA Change Gene 3  3 (G2) 283 TGC (TGT)¹  4 (G2) 2288 No change³ 747  5 (G3) 1306 AGC (AGT)  6 (G3) 169 ACA (ACG)  9 (G2) 2388 TCA (TTA) 780 Phe (Leu)² 21 (G3) 2388 No change 780 25 (G3) 308 No change 87 29 (G1) 874 TTC (TTA) 38 (G4) 448 GAC (GAT) Gene 4  1 (G1) 1953 TCA (TCC)  6 (G3) 1608 GGC (GGT) 11 (G3) 417 ACG (ACA) 12 (G3) 1481 ACA (AGA) 501 Thr (Arg) 16 (G1) 1953 TCA (TCC) 19 (G2) 1674 No change 20 (G3) 1674 No change Gene 5  6 (G3) 75 AAA (ACA) 22 Lys (Thr) 20 (G3) 75 AAA (ACA) 22 ys (Thr)  3 (G2) 1204 GAA (GAG)  4 (G2) 1204 GAA (GAG)  5 (G3) 394 No change  8 (G2) 667 TTT (TTC)  9 (G2) 667 TTT (TTC) 10 (G3) 1186 TAC (TAT) 29 (G1) 1219 ACG (ACA) Gene 9 22 (G1) 556 ATA (GTA) 170 Ile (Val) 29 (G1) 556 ATA (GTA) 170 Ile (Val) 37 (G4) 263 CGA (CAA) 72 Arg (Gln) Gene 10 22 (G1) 92 ATA (ATG) 17 Ile (Met) 22 (G1) 174 ACA (GCA) 45 Thr (Ala) 22 (G1) 218 AAG (AAA) ¹Underlined nucleotide is different in the clinical isolate when compared to the Manufacture's Working Virus Seed (MWVS). Triplet in parentheses is the consensus sequence of MWVS. ²Amino acid (aa) in parentheses is present in the relevant MWVS. ³Initial clonal sequence analysis had indicated heterogeneity at the positions listed as “No change”. Conseusus sequence analysis of these isolates revealed sequence identity with the corresponding MWVS. Boldface type indicates that the nucleotide change was identified in more than one clinical isolate. shown in Table 9. Analysis of the micro-heterogeneity present in the commercial seeds at nucleotide position 174 in gene 10 revealed 25% and 23% of the minor species (A) in the G1 and G4 viruses, respectively, as shown in Table 10; these were similar to the levels observed in the clinical virus seed bank (12% and 7% respectively). As observed in the clinical seeds, the RRV G3 commercial seed strain (MWVS-7) contained solely A at nucleotide position 174. The G2 strain of the commercial seed bank (MWVS-6), however, did not retain the same minor population observed in the G2 clinical strain (5% G), but instead, resembled the G3 RRV strain at this position, harboring 100% A at this site by clonal analysis.

Determination of the heterogeneity at nucleotides 2388 and 174 of genes 3 and 10, respectively, in several vaccine lots produced from the commercial manufacturer's working virus seed allowed the monitoring of the genetic stability of these positions after passage in vitro. Four vaccine lots of each serotype, generated from the commercial virus seeds (MWVS-5, 6, 7 and 8) were analyzed by clonal analysis. Four G2 vaccine lots (I973029, I983008, I983037 and I973030) were analyzed for heterogeneity at nucleotide position 2388 in gene 3, and in each case, the level of the minor variant (4%, 8%, 8% and 16% C) was found to be similar to the 15% observed in the G2 commercial seed (MWVS-6) (Table 9). For nucleotide 174 in gene 10, each of the four G1 and G4 vaccine lots contained the minor species (A) at a level similar to that seen in its corresponding commercial seed. The four G1 vaccine lots (I973020, I973017, I983026 and I983003) contained 22%, 6%, 33% and 4% A, respectively, at nucleotide position 174 compared to the 25% A observed in the G1 commercial (MWVS-5) seed. Likewise, the G4 vaccine lots (I973034, I973004, I973030 and I983030) retained 9%, 14%, 19% and 23% A, respectively, at this position, similar to the 23% A found in the G4 (MWVS-8) seed (Table 10). These data indicate that conditions used in the vaccine manufacturing process preserve the identity of the four RRV vaccine strains as reflected in their genomic nucleotide sequence.

TABLE 9 Base Composition at Nucleotide 2388 in Gene 3 for Serotype 2 Manufacturer's Working Virus Seed and Vaccine Concentrates Nucleotide representing consensus/variant (% population) Manufacturer's Working Virus Seed - Commercial RRV Commercial Vaccine Concentrates and Harvests Serotype (MWVS-6) Lot #I973030 Lot #I973029 Lot #I983008 Lot #I983037 2 T/C (85/15) T/C (84/16) T/C (96/4) T/C (92/8) T/C (92/8)

TABLE 10 Base Composition at Nucleotide 174 in Gene 10 for Manufacturer's Working Virus Seeds and Vaccine Concentrates Nucleotide Representing Consensus/Variant (% Population) Manufacturer's Working Virus Serotype Seeds RRV Commercial Vaccine Concentrates and Harvests* 1 G/A (75/25)²⁴ G/A (78/22)²³ G/A (94/6)³² G/A (67/33)²⁴ G/A (96/4)²⁴ 2 A/G (100/0)²⁹ A/G (100/0)²⁶ A/G (100/0)²¹ A/G (100/0)²¹ A/G (100/0)²⁴ 3 A/G (100/0)³⁰ A/G (100/0)⁴³ A/G (100/0)²³ A/G (100/0)³⁷ Not Determined 4 G/A (77/23)³⁰ G/A (91/9)²³ G/A (86/14)²⁸ G/A (81/19)²¹ G/A (77/23)⁷¹ Manufacturer's Working Virus Seeds = MWVS-5, 6, 7 and 8 for Serotypes 1, 2, 3 and 4, respectively. *Vaccine Concentrates and Harvests: Serotype 1 - Lot # I973020, I973017, I983026 and I983003, respectively. Serotype 2 - Lot # I973030, I973029, I983008 and I983037, respectively. Serotype 3 - Lot # I973026, I983021 and I983032, respectively. Serotype 4 - Lot # I973034, I973004, I973040 and I983030, respectively. Superscripts denote the number of clones sequenced for each virus lot

Example 3 Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC)

A sensitive and direct method to monitor the levels of micro-heterogeneity at nucleotide 2388 of gene 3, Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC), was developed.

Materials

The following materials were used: Life Technologies (Gibco-BRL) SuperScript™ Preamplification for First Strand cDNA Synthesis kit; RNase-free water; RRV Serotype 2 total RNA; Gene 3 nucleotide 2388 100% T DNA control template (1/30 dilution of stock); Gene 3 nucleotide 2388 100% C DNA control template (1/30 dilution of stock); Perkin Elmer Thermal Cycler PE 480; primer RRV-G3-EcoRI (20 pmole/μl); primer RRV-302A-flourescein (20 pmole/μl); Perkin-Elmer Taq DNA polymerase; mineral oil (Sigma), molecular biology grade 5; Perkin-Elmer 0.5 ml Gene-Amp reaction tubes; Pharmacia G-40 AutoSeq spin columns; restriction endonuclease Eco RI (10 U/μl) (Roche Molecular Biochemicals); glycerol; bromophenol blue; xylene cyanol; Bio-Rad 40% acrylamide:bis-acrylamide (38:2) liquid; distilled water; 10×TBE (Gibco-BRL); TEMED; ammonium persulfate; two 20×20 cm vertical polyacrylamide gel electrophoresis apparatus with 0.75 mm, 20-well combs and 0.75 mm spacers; flat head gel loading pipet tips; Molecular Dynamics Flourimager 595.

Reverse Transcription

For each sample, 500-1000 ng RNA and water were added to a 0.5 μl microfuge tube for a final volume of 11 μl. It was preferable to use 1000 ng RNA per reaction if the RNA is concentrated enough; however, 500 ng per reaction was usually sufficient to generate product. The RNA and water mixture was heated in a preheated thermal cycler at 96° C. for 4 minutes, and then immediately placed on ice for 2-3 minutes. During that time, the RT mix was made as follows:

10X PCR Buffer (Superscript II kit) 2 μl 25 mM Magnesium chloride (Superscript II kit) 2 μl 10 mM dNTP mix (Superscript II kit) 1 μl 0.1 M DTT (Superscript II kit) 2 μl RV-302A-Flouroscein primer (20 pmole/μl) 1 μl TOTAL: 8 μl.

After cooling, the chilled RNA and water mixture was briefly centrifuged to spin down any condensation on the tube cap. Then 8 μl RT mix (above) was added to each tube of RNA and water mixture, and mixed by pipeting up and down several times. A drop of mineral oil was overlaid in the reaction tube, and the tube was then incubated in a pre-heated thermal cycler at 50° C. for 5 minutes. Subsequently, 1 μl Superscript II Reverse Transcriptase 200 U/μl (kit) was added to each reaction tube underneath the oil layer. The reverse transcription process was allowed to proceed at 50° C., for 60 minutes, followed by 70° C. for 15 minutes to inactivate the RT; the mixture was then soaked at 4° C. One μl RNase H (Superscript II kit) was added to each reaction underneath the oil, and the reaction was incubated in a thermal cycler at 37° C. for 20 minutes. The first-strand cDNA resulting from this procedure could either be transferred to 4° C. and used directly in the PCR reaction, or stored at −20° C.

PCR Step

It should be noted that in addition to the vaccine samples, each assay must be accompanied by two DNA control reactions containing either 100% T or 100% C DNA templates, to validate each assay.

PCR mix was prepared as follows:

10X PCR Buffer (Superscript II kit)   5 μl 25 mM magnesium chloride (Superscript II kit)   3 μl 10 mM dNTP mix (Superscript II kit)   1 μl RRV-302A-Flouroscein primer (20 pmole/μl) 1.5 μl RRV-G3-EcoRI primer (20 pmole/μl) 1.5 μl Taq DNA polymerase (Perkin-Elmer) 0.5 μl Nuclease-free water  35 μl TOTAL: 47.5 μl. 

The PCR mix was aliquoted to a fresh 0.5 ml tube for each sample. Three μl of the first strand cDNA (from the RT reaction), 3 μl of 1/30 dilution of 100% T DNA, or 3 μl of 1/40 dilution of 100% C DNA were added to each PCR reaction as a template, and the reaction was then overlaid with a drop of mineral oil. The reaction was placed in a pre-heated thermal cycler, and cycled for 94° C. for 1 minutes (1 cycle), followed by 94° C. for 30 seconds and 60° C. for 3 minutes (40 cycles), and then a 4° C. soak.

Since the PCR products being generated are fluorescent, their exposure to light should be minimized by placing a piece of aluminum foil over the thermal cycler cover during cycling. Storage of the PCR products should always be in light tight containers.

Purification and Digestion of PCR Products

A Pharmacia G-40 spin column was prepared for use in the purification of each PCR product. Each column was vortexed for 2-3 seconds to thoroughly resuspend the Sephadex beads; the screw cap was loosened one-half turn; the bottom of the column was snapped off and discarded, and then the screw cap was removed and discarded as well. Each column was placed in an empty 1.5 ml Eppendorf tube and spun in an Eppendorf microfuge at 3200 rpm (approximately 200×g) for 1 minute. Columns were then used immediately to avoid drying of the resin. The PCR reaction described above was removed from each tube, being careful to transfer as little oil as possible. The G-50 spin column was removed from its tube, and the entire 50 μl PCR reaction was slowly loaded onto the center of the angled resin in the column. The loaded column was then placed into a fresh, labeled 1.5 ml Eppendorf microfuge tube. The tubes were spun at 3200 rpm for 1 minute to collect the effluent containing the purified PCR product, and column was discarded. This purification step allows subsequent digestion of the PCR product with EcoRI to take place in a proper restriction enzyme buffer, as digestion with EcoRI in other buffers results in non-specific digestion of PCR products. Eight μl of each purified PCR reaction was removed to a new 0.5 ml Gene-amp tube, and 1 μl of Restriction Buffer H (Roche Mol. Biochemicals) provided with the EcoRI enzyme was added. One μl of EcoRI restriction enzyme (10 U/μl) was added to each sample and pipetted up and down several times to thoroughly mix reaction contents. The reaction tubes were then placed in a thermal cycler at 37° C. for 3-4 hours. The reactions were spun down by pulsing in microfuge to spin down any condensation on the tube cap, and 2 μl of 6× loading dye (40% glycerol, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol) were added to each digested sample, and then the samples were stored at 4° C. in light tight containers until loaded onto the gel.

Polyacrylamide Gel Electrophoresis of Digested PCR Products

During the final hour of the digestion step (above), polyacrylamide gels were prepared for analyses of the digested PCR products. Gel plates were washed with Alconox detergent, followed by a final rinse with ethanol, and allowed to air dry. The plates were assembled, and a 6% non-denaturing polyacrylamide gel mixture was prepared as follows:

Distilled water 45 ml 10X TBE  6 ml 40% acrylamide:bis (38:2)  9 ml TOTAL: 60 ml (This gel recipe was sufficient to pour two 20×20 cm gels; each gel accommodated nine samples.)

For polymerization, 50 μl TEMED and 500 μl freshly prepared 10% ammonium persulfate were added to the gel mixture, swirling gently to mix reagents. The gel was immediately poured, the comb inserted, and clamps placed on the wells. Polymerization was allowed to occur for approximately one hour, after which the comb was removed and the wells rinsed with 1×TBE. The bottom buffer chamber was filled with 1×TBE. An entire 12 μl of each sample was loaded onto the gel, and the gel was run at 200-220 volts until the xylene cyanol was approximately 2 cm from the bottom of the gel (approximately 3 hours).

Quantitation of Undigested and Digested Band Density by Flourimaging

The gels were transferred to an overhead transparency, and then to a glass sample plate on the Flourimager. The gel was scanned on the Flourimager with the following settings: voltage (PMI)=600; filter 1=530 dF30 agarose; wavelength=488 in. Once the gel was scanned, the image could be modified and quantitated using ImageQuant2 software. The percent C at nucleotide 2388 was then calculated:

-   a) Corrected volume of undigested product (CU)=undigested band     volume−background volume. -   b) Corrected volume of digested product (CD)=digested band     volume−background volume. -   c) Percent 2388 “C”=[CD/(CD+CU)]×100.     To ensure validity of each individual determination, all of the     following conditions were met: 1) the value of % 2388 C in the 100%     C DNA control sample must have been ≧905; 2) the value of % 2388 C     in the 100% T DNA control sample must have been less than 1.5%;     and 3) the total bands of fluorescence (indicated by the volume)     present in the undigested and digested bands must have been     ≧500,000.

Results

RNA was extracted as described above from each of 15 serotype G2 commercial vaccine lots, and for each vaccine lot, 5 individual determinations of the level of variant “C” at nucleotide position 2388 were carried out. The RNA from each virus sample was used to synthesize first strand cDNA (reverse transcription) in two independent experiments carried out on separate days. Three independent determinations of % C at nucleotide position 2388 were made using cDNA derived from the first reverse transcription reaction as PCR template, while the remaining two independent determinations were carried out using cDNA derived from the second reverse transcription reaction. For each sample, the Mean and Standard Deviation of the five determinations was calculated.

Following the protocols described above, a short region of gene 3 of RRV serotype G2 virus, encompassing nucleotide position 2388, was amplified by RT/PCR using the Superscript Pre-amplification System for First Strand cDNA synthesis (Life Technologies, Rockville, Md.). One of the primers used for amplification was homologous to the sequence immediately upstream of nucleotide 2388 and was designed to create an Eco RI restriction enzyme site if a cytosine residue was present at position 2388 (RRV-G3-EcoRI primer, GTTAGTGGAGTTCTAGCGACATATTTTAAAATGTAGAAT (SEQ ID NO: 186), corresponding to plus sense nucleotides 2347-2386). The second primer (RRV-302A-5′Flourescein, GGTCACATCATGACTAGTGTG (SEQ ID NO: 187), corresponding to negative sense nucleotides 2571-2591) was labeled with Flourescein (at the 5′ end), enabling the resulting PCR product to be visualized and quantitated using polyacrylamide gel electrophoresis and flourimaging techniques.

Following amplification, the PCR product was purified and digested. PCR products derived from genomes containing cytidine (C) at nucleotide position 2388 were digested, resulting in 34 and 207 bp digested fragments; of these two fragments, only the 207 bp fragment retains the flourescein tag and was detected by PAGE). Those genomes containing thymidine (T) at nucleotide position 2388 did not generate an EcoRI site, and thus yielded a 244 bp uncleaved product. The products were separated on a 6% polyacrylamide gel, and the relative densities of bands representing the undigested and digested products were quantitated using the Flourimager 595 (Molecular Dynamics), and a measurement of background fluorescence was taken. The percentage of viral genomes in the sample containing “C” at nucleotide 2388 was then calculated.

Base mixtures at nucleotide position 2388 in gene 3 and nucleotide position 174 in gene 10 were consistently detected in vaccine virus at the master working virus seed (MWVS) and vaccine monopool bulk concentrate stages. Thus, measurement of the inherent heterogeneity at these positions in the vaccine virus lots can be used as the foundation for a product consistency assay. The MAPREC analysis was used to determine the base composition at nucleotide 2388. The low level of variation observed in the fifteen G2 commercial vaccine lots at this nucleotide position demonstrates, not only the stability of the subpopulation containing C at nucleotide, but also the consistency of the vaccine manufacturing process. The average percentage of C at nucleotide position 2388 ranged from 2/75% to 6.68%, resulting in a variation of no more than 3.92% when any of the fifteen vaccine lots were compared (Table 11). The first three MAPREC determinations, revealed slightly higher levels of the minor species (C) compared to the remaining twelve G2 vaccine lots. However, three additional determinations carried out from a new RT reaction yielded levels of C consistent with those observed in the other twelve lots. Reanalyzing the data to exclude the first three MAPREC determinations, the range of variation observed for the fifteen vaccine lots was significantly reduced (i.e., a low of 2.76% and a high of 4.90%, equivalent to a variation of just 2.14%). In each case, excluding the three aberrant determinations, the standard deviation observed across five MAPREC determinations of the percentage C at nucleotide position 2388 of gene 3 in the commercial vaccine lots fell well below 1%, with over 50% of the samples resulting in standard deviations less than 0.5%. Taking into account that the variation observed was nearly 4 times greater by clonal analyses

TABLE 11 MAPREC Analysis of % C at Nucleotide Position 2388 in RRV Gene 3 Sample Standard Name Trial#1 Trial#2 Trial#3 Trial#4 {circumflex over ( )}Trial#5 *Trial#5B {circumflex over ( )}Trial#6 Average Duration 100% T- 1.13 1.04 0.85 0.47 0.86 0.87 0.25 DNA Control 100% C- 92.24 93.38 93.85 93.94 93.33 93.35 0.68 DNA Control Serotype 3.60 4.16 3.56 4.88 4.20 3.12 3.11 3.80 0.64 2-I97328 Serotype 3.38 3.08 4.16 5.20 4.97 3.39 3.17 3.91 0.88 2-I973029 Serotype 4.06 3.11 4.72 3.38 4.62 4.04 3.38 3.90 0.63 2-I973030 Serotype 4.16 4.60 3.82 3.32 3.23 2.58 3.76 3.64 0.66 2-I973032 Serotype 7.69 5.38 12.31 4.97 5.41 4.34 6.68 2.98 2-I983005 Serotype 6.67 4.33 9.00 4.12 5.25 5.11 5.75 1.83 2-I983006 Serotype 6.68 5.20 6.11 4.14 4.04 4.31 5.08 1.11 2-I983007 Serotype 2.97 3.97 3.46 2.79 2.85 3.65 3.28 0.48 2-I983008 Serotype 3.76 3.72 3.63 3.06 3.46 3.60 3.54 0.26 2-I983009 Serotype 3.65 4.36 3.83 4.01 4.13 3.81 3.97 0.26 2-I983011 Serotype 3.14 3.52 3.30 2.82 2.95 2.76 3.08 0.29 2-I983012 Serotype 3.32 3.99 3.55 3.48 2.55 3.72 3.44 0.49 2-I983013 Serotype 3.36 3.35 3.82 3.83 3.97 3.67 0.29 2-I983037 Serotype 4.16 4.67 4.76 4.05 4.56 4.44 0.32 2-I983038 Serotype 4.88 3.75 3.84 3.38 4.49 4.07 0.61 2-I983039 *A second digestion sample taken from the PCR rxn generated in trial 5. (A digestion duplicate) {circumflex over ( )}Trials 5 and 6 were PCR rxns. seeded from the same RT rxn. and carried out on the same day. than was found using MAPREC (8% vs 2.14%, respectively), the levels of the minor species (C) in the G2 vaccine pools as measured by either method are comparable (average of 4% for MAPREC vs 8% for clonal analysis). These data indicate that MAPREC analyses of base composition at nucleotide 2388 in gene 3 of RRV were both accurate and reproducible.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. 

1. An isolated nucleic acid molecule having a sequence selected from the group consisting of: SEQ ID NO:1-14, inclusive.
 2. An isolated nucleic acid molecule encoding a polypeptide having a sequence selected from the group consisting of: SEQ ID NO: 15-28, inclusive.
 3. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 1 and having a nucleotide change to a G at nucleotide 2120
 4. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 2 and having one or more of the following nucleotide changes: a C at nucleotide 493, and a T at nucleotide
 947. 5. An isolated nucleic acid molecule having the sequence of SEQ ID NO:3 and having one or more of the following nucleotide changes: an A at nucleotide 169, a C at nucleotide 283, a C at nucleotide 448, a C at nucleotide 874, a C at nucleotide 1306, and a C at nucleotide
 2388. 6. An isolated nucleic acid molecule having the sequence of SEQ ID NO:4 and having one or more of the following nucleotide changes: a C at nucleotide 119, a G at nucleotide 417, a G at nucleotide 809, a C at nucleotide 977, an A at nucleotide 1463, a C at nucleotide 1481, a C at nucleotide 1608, a C at nucleotide 1755, and an A at nucleotide
 1953. 7. An isolated nucleic acid molecule having the sequence of SEQ ID NO:5 and having one or more of the following nucleotide changes: an A at nucleotide 75, a T at nucleotide 84, a C at nucleotide 347, a T at nucleotide 667, a C at nucleotide 1186, a G at nucleotide 1219, and an A at nucleotide
 1204. 8. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 6 and having one or more of the following nucleotide changes: a C at nucleotide 376, an A at nucleotide 756, an A at nucleotide 1008, and a G at nucleotide
 1041. 9. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 7 and having a nucleotide change to a G at nucleotide
 387. 10. An isolated nucleic acid molecule having the sequence of SEQ ID NO:10 and having one or more of the following nucleotide changes: an A at nucleotide 92, an A at nucleotide 174, and a G at nucleotide
 218. 11. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 11 and having a nucleotide change to A at nucleotide
 180. 12. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 12 and having a nucleotide change to an A at nucleotide
 556. 13. An isolated nucleic acid molecule having the sequence of SEQ ID NO: 14 and having a nucleotide change to a G at nucleotide
 263. 14. A vector comprising an isolated nucleic acid molecule of claim
 1. 15. A recombinant host cell comprising a vector of claim
 14. 16. A method for producing a polypeptide encoded by an isolated nucleic acid molecule, comprising culturing the recombinant host cell of claim 15 under conditions suitable for expression of said nucleic acid molecule. 